Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

Abstract

Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.

Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

“…However, under NMB and red light treatment, the most stable reference genes were ftsZ, proC, and fabD, as indicated by the 3 software packages. Identical observations were demonstrated by DeLorenzo and Moon 41 . Unfortunately, according to the literature, there were some discrepancies in the results obtained by BestKeeper, geNorm and NormFinder software 36,39,40 .…”

“…The 16S rRNA gene has also been proven unsuitable for the analysis of iron-regulated gene expression in Pseudomonas brassicacearum (it ranked sixth of the eight genes tested) 40 . On the other hand, based on mathematical models (BestKeeper, geNorm and NormFinder software), 16S rRNA was classified as one of the best reference genes to evaluate expression levels in Rhodococcus opacus under different growth conditions 41 . It should be noted that 16S rRNA may not be suitable for analyses in which the ability to detect nonviable and dead bacterial cells interferes with results 42 .…”

Identification of Resistance Genes and Response to Arsenic in Rhodococcus aetherivorans BCP1

“…RT-qPCR experiments were performed in triplicate and negative controls were performed by omitting the reverse transcriptase to check for RNA purity from genomic DNA. The expression levels of target genes were normalized to the expression level of the reference gene 16S rRNA, which was previously reported as a stable reference gene in a Rhodococcus strain (DeLorenzo and Moon, 2018). The fold changes for each target genes were obtained using the ΔΔCt method (Pfaffl, 2001) considering the growth on glucose as the reference condition.…”

Functional Genomics Insights Into the Pathogenicity, Habitat Fitness, and Mechanisms Modifying Plant Development of Rhodococcus sp. PBTS1 and PBTS2

“…The primer sequences used for the detection of vicA, vicA2, 16S rRNA, ITS, fasC, fasD, fasE, and fasF either by quantitative (q)PCR or endpoint PCR are given in Supplementary Table S1 (Nikolaeva et al, 2012;Dhandapani et al, 2017;DeLorenzo and Moon, 2018).…”